Chondral defects refer to focal lesions or damage in the articular cartilage, which are difficult to treat. If left untreated, these abnormalities can lead to an increased risk of osteoarthritis (OA), a primary chronic joint disease caused by various factors. The worldwide prevalence of OA is estimated to be around 250 million people. This painful condition is characterized by symptoms such as stiffness, muscle loss, fatigue, insomnia, stress, depression, stigmatization, and a decline in quality of life (1). In the field of orthopaedics, several treatment options have been developed to manage these anomalies. Osteoarticular transplants have emerged as the first and most common procedure, followed by the gold standard microfracture procedure that has been widely used for nearly 30 years. In the late 1990s, the implantation of autologous chondrocytes was introduced as an alternative treatment. Scaffold-based techniques entered the scene in the early 2000s (2). While these therapies offer advantages, they also come with drawbacks such as the need for open surgery, primary chondrocyte dedifferentiation, a lengthy recovery period, and morbidity at the donor site. As a result, the treatment of cartilage abnormalities remains challenging. However, there is hope in contemporary cell-based therapy utilizing mesenchymal stem cells (MSCs). This approach has significant implications, such as immunological rejection and tumorigenicity. Another promising alternative to cell-based therapy is exosome-based cell-free therapy (3).

Exosomes, the smallest vesicles ranging from 30 to 150 nm in diameter, are released from cells and play a crucial role in cell-to-cell communication. These vesicles carry a cargo of proteins, mRNA, DNA, RNA, and lipids, and recent evidence suggests that they are nature’s delivery system. Exosomes are produced by endosomes and can be released by cells in both physiological and pathological conditions. Research has shown the involvement of exosomes in cartilage-associated defects (4). Exosomes derived from OA joints have been found to carry pathogenic signals, including proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, and IL-22. These cytokines are believed to contribute to the degradation of the extracellular matrix (ECM) by attracting matrix metalloproteinases (MMPs) (5). Exosomes derived from primary chondrocytes, when combined with hydrogels, have shown successful outcomes in chondral healing by delivering sustained release of exosomes to the target location and enhancing chondrocyte proliferation, migration, and matrix production (6). In addition, exosome therapy has been shown to promote the formation of hyaline cartilage by injecting exosomes directly into the affected joint. This treatment has been found to significantly regenerate cartilage lesions in a model of osteochondral defects (7).

This review article delves into the potential of exosomes in treating disorders associated with cartilage, with a particular focus on OA. The article provides an overview of exosomes and their role in intercellular communication, followed by a discussion of their use in diverse ailments, core focus on OA. It also explores the mechanism of action of exosomes with native cells and their clinical applications. Furthermore, the article provides updates on clinical trials involving exosomes for different conditions. Lastly, the article concludes with a comprehensive discussion on the utilization of exosomes in the context of OA.

Extracellular vesicles

Extracellular vesicles (EVs) play a crucial role in cell-cell communication, migration, and various biological processes. Cells release EVs in both normal and pathological conditions, including exosomes, microvesicles, and apoptotic bodies predominantly (Fig. 1). However, recent studies have also identified other types of EVs such as migrasomes, exophers, exomeres, supremeres, chromatimeres, lipoproteins, oncosomes, and others. Each type of vesicle has its distinct origin and function (Table 1). The nomenclature of vesicles has previously puzzled researchers since all vesicles are secreted by cells. EVs carry a cargo of proteins, mRNA, lipids, and DNA (8). They can be classified based on their sedimentation properties and composition. On average, EVs have a sedimentation rate ranging from 1.10 to 1.30 g/ml. Exosomes are characterized by specific markers such as CD63, CD9, CD81, Alix, and Tsg101. Microvesicles, on the other hand, are distinguished by the presence of selectins and integrins, while apoptotic bodies contain histones and DNA. In terms of lipid content, microvesicles have an exposed phosphatidylserine, whereas exosomes have an exposure. The roles in biological activities encompass waste disposal, protein and RNA transfer for functionality, and intercellular communication. Each extracellular vesicle has distinct molecular features due to its specific biogenesis mechanism. Exosomes represent a subgroup of EVs, typically measuring 30∼150 nm in diameter and having a density of 1.13∼1.19 g/ml. Exosomes serve as disease indicators, biomarkers, and therapeutic targets. They possess the ability to directly target cells, regulate the microenvironment and inflammation, and promote tissue regeneration. In both normal and pathological situations, they play a crucial role by facilitating cell-to-cell communication. Exosomes were initially discovered in the 1980s as endocytic microvesicles released by maturing reticulocytes by Pan and Johnstone. While Trams’ publication was frequently referenced during the 1980s, the term “exosome” did not resurface to refer to EVs until 1986 and again in 1987, coined by Rose Johnstone. Although the name “exosome” for these EVs was later adopted by Rose Johnstone, the term had been previously used to describe other membrane fragments isolated from biological fluids. Exosomes were initially regarded as cellular “trash bags,” but it wasn’t until the mid-1990s that their vital roles in intercellular communication in normal physiological processes and disease pathogenesis, including cancer, were gradually revealed (9). Nearly all cells release exosomes, with their biogenesis commencing at the cell surface.

Table 1 . Hallmarks of extracellular vesicles

Characteristics	Exosomes	Microvesicles	Apoptotic bodies	Exophers	Migrasomes	Exomeres	Supremeres	Chromatimeres	Lipoproteins	Oncosomes
Size	30∼150 nm	100∼1,000 nm	0.5∼5,000 nm	3.5∼4 μm	<4 μm	≤50 nm	N/A	N/A	∼30 to 150 nm HDL (5∼15 nm)	1∼100 μm
Shape	Spherical/cup shaped	Irregular	Irregular	Irregularly shaped but are typically spherical structures	Oval shaped	N/A	N/A	N/A	Lipoprotein-like structures, micelle-like structures	Cup-shaped vesicles
Density	1.13∼1.19 g/ml	1.25∼1.30 g/ml	1.16∼1.28 g/ml	N/A	N/A	Lower density	Vary depending on their buoyant density	N/A	0.930∼1.210 g/ml	Discrete buoyant densities
Sedimentation rate	100,000∼200,000 ×g	10,000∼20,000 ×g	1,200, 10,000, or 100,000 ×g	N/A	N/A	N/A	N/A	N/A	N/A	N/A
Origin	Multivesicular bodies (endocytic pathway)	Plasma membrane	Various cell types	Evagination of the cell membrane	Tetraspanin- enriched macrodomain accumulation	N/A	N/A	N/A	Synthesized primarily in the liver	Cancer cells
Mechanism of release	Exocytosis (MVBs) (inward budding)	Outward budding of plasma membrane	Cell death causes cell shrinkage and blebbing of the plasma membrane	Pinching-off mechanism	Released from the retraction fibers during cell migration	N/A	N/A	N/A	Endogenous lipoprotein pathway	Shedding of plasma membrane blebs
Content	mRNA, miRNA, proteins, lipids	mRNA, miRNA, proteins, lipids	Proteins, nuclear segments, DNA, RNA, lipid cellular debris	Organelles, large protein complexes, aggregated, soluble proteins, and other cytoplasmic components	mRNA, protein, or damaged mitochondria, or as chemoattractive sources	Proteins, nucleic acids and lipids	N/A	DNA	Cholesterol	Distinct protein cargo, tumor DNA
Biomarkers	Exosomal markers- ALIX, TSG101, HSC70, CD63, CD9, CD81, and HSP90	Selectins, integrins, CD40	Histones, HSP60, GRP78	N/A	N/A	N/A	N/A	N/A	Apolipoprotein B, sphingolipids/ ceramides	Cancer-specific biomarkers
Lipid composition	Cholesterol, sphingomyelin and ceramide-rich lipid rafts, low phosphatidylserine exposure	High phosphatidylserine exposure, cholesterol	N/A	Unknown lipid bilayer composition	N/A	Lipid bilayer membrane	N/A	N/A	Saturated and monoenoic fatty acids	N/A
Biological purpose	Cell-to-cell communication, migration, and maintenance, as well as a payload of proteins, DNA, and RNAs that imitate the parent cell	Role in intercellular communication	Efficient removal of cell debris	Related to autophagy	Cell migration	Intercellular communication	Large protein complexes, including ribosomes and proteasomes	Complex of DNA and proteins	Transport of lipids, immunomodulation	Oncogenic transformation, intercellular communication
Pathway	ESCRT- dependent and ESCRT– independent, constitutive dependent, stimuli dependent	Constitutive dependent, stimuli dependent Ca2 dependent	Apoptosis dependent	Ubiquitin- proteasome system and autophagy- lysosome pathway	N/A	N/A	N/A	N/A	Exogenous and endogenous lipoprotein pathway	AKT1 and EGFR pathway, c-MET pathway
Quantification	DLS Nanoparticle tracking analysis TEM, SEM	N/A	N/A	No standard methodologies	N/A	N/A	N/A	N/A	FRET-based assay, tunable resistive pulse sensing, flow cytometry	FRET-based assay, DLS
Isolation methods	Ultracentrifugation Size exclusion chromatography Tangential flow filtration EXO-Kit methods	No standard methodologies	Ultracentrifugation	No standard methodologies	N/A	Ultracentrifugation and asymmetric- flow field-flow fractionation	Ultracentri-fugation, density gradient centrifugation	N/A	Ultracentrifugation, size exclusion chromatography	Physicochemical methods, ultracentrifugation

N/A: not available, HDL: high density lipoprotein, MVBs: multi-vesicular bodies, ESCRT: endosomal sorting complex required for transport, DLS: dynamic light scattering.

Figure 1. Biogenesis of extracellular vesicles. Schematic representation of the biogenesis of exosomes starts with double invagination of the plasma membrane. Further, formation of multi-vesicular bodies (MVBs) and followed by the exocytosis. Exosome secretion and composition (endosomal sorting complex required for transport [ESCRT] dependent). MVBs arise as buds from the plasma membrane during inflammatory and hypoxic circumstances. Apoptotic blebs are extracellular vesicles (EVs) that are produced in response to increased cell contraction and hydrostatic pressure. Only during planned cell death are apoptotic bodies released. Exomeres are newly found EVs with unknown biological functions and biogenesis. Migrasomes are oval-shaped EVs that are generated during cell migration. Oncosomes are big and tiny EVs produced from the membrane that are discharged by cancer cells. They have a distinct signature from the tumor cells from which they are released.

Definition of Exosomes

Biogenesis of exosomes: Exosomes are membrane-bound endosomes-derived vesicles that begin with a twofold invagination of the plasma membrane. Later on, the cargoes enter, forming multi-vesicular bodies (MVBs). Intraluminal vesicles are exosome precursors formed by inward budding of MVBs (8). The biogenesis and release of exosomes are regulated by various factors, such as endosomal sorting complex required for transport (ESCRT), which consist of ESCRT proteins (ESCRT-0, I, II, and III) and accessory proteins like ALIX, vacuolar protein sorting-associated protein 4, Rab GTPase activating protein, sphingomyelinase, and ceramide. Additionally, mechanistic target of rapamycin complex 1 (mTORC1) regulates exosome release in response to changes in nutritional and growth factor conditions, and that mTORC1 activation limits exosome release in cultured cells and in vivo (10). Lysosomes combine with lipid-deficient MVBs for destruction. Once exosomes are released, they can interact with adjacent cells and initiate various biological processes. Their unique contents, including their binding to ECM components like fibronectin and collagen, play a role in defining their characteristics.

Isolation of exosomes: Exosomes can be isolated using ultracentrifugation, size exclusion chromatography, tangential flow filtration, coprecipitation, and immunoaffinity techniques. However, ultracentrifugation is time-consuming and results in a relatively low exosome yield. Instead, chromatography-based approaches have been adopted to isolate exosomes based on size. Gel selection is essential for exosome isolation, as denser compounds have short pathways in the gel, while smaller vesicles have a long retention period. Exosomes were created in a conditioned medium with tangential flow filtration to size fractionate the medium. Tangential flow filtration was used to create exosomes in a conditioned medium, ensuring effective ultra-centrifugation. Small extracellular vesicles (sEVs) were extracted using size exclusion chromatography. During low-speed centrifugation, polyethylene glycol (PEG) hydrophilic polymers interacted with exosomes, reducing their solubility and precipitating them (11). Researchers have also developed novel methods for isolating exosomes using affinity capture with antibodies. Filipović et al. (12), used single domain antibodies that had been chosen from the naive library and confirmed to have exosome stabilizing properties. However, binding to the targets was complicated due to specialized antibodies. This approach effectively isolated vesicles with exosome-like morphology and biochemistry. Exosomes have been found to play a crucial role in intercellular communication and hold promise for ther-

apeutic applications in disorders such as cancer, inflammation, and neurological diseases. Immunoaffinity interactions provide an intriguing method for exosome separation, although it is a laborious and costly process. How-ever, there is no single optimal method for exosome isolation, and an inclusive approach using multiple techniques may create the best results. Following the Minimal Information for Studies of Extracellular Vesicles guidelines of 2018, specificity and recovery are the main variables of each isolation technique (13).

Characterization of exosomes: Identification of exosomes after isolation is crucial for understanding their properties. Various methods, such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), western blot, and flow cytometry, can be used for this purpose. TEM is commonly employed to identify and characterize a single exosome, as it reveals the typical cup-shaped structure. SEM utilizes the collection of ejected electrons to produce images. NTA, on the other hand, is a light scattering technique that provides information about the size distribution of EVs. Western blotting is a frequently used analysis method due to its simplicity and wide availability in exosome research. Additionally, flow cytometry is another preferred technique for exosome counting, as it can identify exosomes and count particles larger than 500 nm (14). Considering the growing interest in exosome research, it is crucial to have a comprehensive article that highlights the different methods for their isolation and detection. Overall, characterizing exosomes is vital for understanding their properties and exploring potential therapeutic applications.

Exosomes and its impact in cartilage regeneration

Exosomes from the injured region exacerbate damage, while therapeutic cells preserve equilibrium. Exosomes could be extracted from a variety of cell origins; this section will review exosomes from various parent cells and their involvement in regeneration. Exosomes from MSC can enhance cartilage and subchondral bone healing. Bone marrow mesenchymal stem cell (BMSC) derived exosomes enhance the immunological microenvironment by healing IL-1β damaged chondrocytes. Chen et al. (15), examined into the ability of BMSC-derived exosomes to repair mitochondrial malfunction and oxidative stress in degenerative cartilage. Exosomes generated from human adipose derived stem cell (hADSC) isolated miR375-overexpressing exosomes displayed enhanced osteogenic capacity in human BMSC-target cells in vitro and faster bone growth in rat calvarial deficiencies in vivo when compared to an untreated control group (16). Additionally, macrophage-derived exosomes regulate immunomodulation, osteogenesis, and angiogenesis. Wharton jelly derived mesenchymal stem cells exosomes promote cartilage homeostasis by stimulating chondrocyte proliferation, migration, catabolic activity, and anti-inflammatory macrophage infiltration (17). Recent studies, chondrocyte-derived exosomes boost chondrocyte proliferation and migration while also enhancing matrix formation, macrophage modulation, regulate mitochondrion and immune reactivity in OA (18). Exosomes were found to sustain the expression of type 2 collagen and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) in chondrocytes when treated with human IL-1β. Exosomes play an important role in cartilage healing due to their high concentration of syndecans (SDC) and tetraspanins (TSPN). TSPN6 plays a negative role in exosomal release, encouraging lysosomal degradation of SDC4 and syntenins. Long noncoding RNA (lncRNA) is a regulator of transcriptional and translation level processes, and primary umbilical cord MSC exosomes were shown to upregulate lncRNA H19 in culturing in a mechanical stimulating environment (19). Recent study examined at the effectiveness of MSC exosomes in restorative osteochondral lesions in a therapeutically relevant porcine model. MSC exosomes and hyaluronic acid (HA) were reported to ameliorate functional osteochondral healing when provided at a clinically endurable frequency of three weekly intra-articular injections (20). Finally, exosomes produced from multiple sources have demonstrated significant potential in tissue regeneration and regenerative medicine (Table 2). Exosomes can influence cell proliferation and migration, as well as stimulate tissue repair and regeneration, by transporting bioactive substances such as proteins, lipids, and nucleic acids.

Table 2 . Origin and isolation techniques of exosomes

Exosome cell type/origin	Exosome isolation procedure	Method (advantages/disadvantages)	Effects/characteristics	Reference
BMSC-EXOs	Ultracentrifugation (100,000 ×g @4C for 60 min)	Gold standard method for exosome isolation/time-consuming and requires expensive equipment	Repair mitochondrial dysfunction and oxidative stress in degenerative cartilage supplemented with mitochondrial proteins	(10)
Chondrocytes	Ultracentrifugation (100,000 ×g for 60 min)		Induce chondrocyte proliferation and migration, enhance matrix synthesis. Regulation of macrophage	(13)
UMSC	Ultracentrifugation (110,000 ×g for 70 min)		Promote chondral damage repair by upregulating lncRNA H19 in response to mechanical stimulation	(14)
BMSC-EXOs	Ultracentrifugation (100,000 ×g @4C for 90 min)		Regulate the equilibrium by aiding the healing of IL-1β-damaged chondrocytes	(29)
Human-WJMSC	Size exclusion chromatography	High specificity and purity of exosome population/ low yield of exosomes	Transmit particular miRNAs that preserve cartilage homeostasis by stimulating chondrocyte proliferation, migration, catabolic activity, and M2 infiltration	(12)
Human embryonic stem cells E1-MYC 16.3	Tangential flow filtration	Separates exosomes by size and density/time-consuming and requires expensive equipment	Protect the OA joint from damage by promoting cartilage repair, inhibitingsynovitis, and mediating subchondral bone remodeling	(15)

BMSC-EXOs: bone marrow mesenchymal stem cell-derived exosomes, UMSC: umbilical cord derived mesenchymal stem cell, WJMSC: Wharton jelly derived mesenchymal stem cell, lncRNA: long noncoding RNA, IL: interleukin, OA: osteoarthritis.

Exosomes have been affiliated to the development of metabolic diseases and major health problems. Exosomes produced by cancer cells change cell metabolism and act as a potent biomarker. LINC00161, a member of the lncRNA family, was shown to be highly expressed in the serum and exosomes of hepatocellular carcinoma patients (21). Several clinical trials, one of which is NCT03830619, have been conducted to investigate the sensitivity and specificity of serum exosome (sEXO) non-coding RNA as a biomarker for lung cancer diagnosis. miR-532-5p, which is connected to cancer risk factors, perhaps used as a biomarker in urological malignancies to distinguish biochemical recurrence and metastasis (22). The combined use of exosomes carried by exogenous MSCs and endogenous gene modification can provide new ideas for the treatment of tumors. Qiu et al. (23), drugs for the treatment of oral squamous cell carcinoma (cabazitaxel, CTX) were incubated with TNF-related apoptosis-inducing ligand (TRAIL) gene-modified MSCs, and exosomes loaded with CTX/TRAIL were successfully purified (MSCT-EXO/CTX). Subsequently, the anticancer effect of MSCT-EXO/CTX was successfully verified in vitro and in vivo. The consolidated modification of therapeutic molecule loading and MSC exosome membrane surface modification has received increasing attention for its potential to enhance tumor tissue targeting. Exosomes generated from human adipose stem cells increase fibroblast migration, collagen production, proliferation in a dose-dependent manner as well as accelerate wound healing. In vivo study of exosomes treated groups reveals a dose-dependent increase in collagen type I content and expression of the contractile protein α-smooth muscle actin at the damaged site (24). Exosomes and chitosan-graft aniline tetramer hydrogels enhanced collagen deposition, vascularization, angiogenesis, and managed diabetic wounds (25). Additionally, MSC exosomes activate wound healing signalling pathways and promote growth factors. Myocardial infraction is an ischemic heart condition that causes severe mortality and morbidity. Recent research suggest that exosomes derived from MSCs have anti-apoptotic effects via miR-210, while macropha-ges produce miR-155-containing exosomes that inhibit fibroblast proliferation and inflammation, leading to impaired heart repair after myocardial infarction (26). Exo-some’s release α-synuclein, which is essential for Parkinson’s disease progression. Alzheimer’s disease releases amyloid β-peptide 1-42 from cells into extracellular space. β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) cleaves APP to produce amyloid peptide, essential for Alzheimer’s disease. Exosomes targeted microglia, neurons, and oligodendrocytes after intravenous administration, lowering BACE1 mRNA and protein levels (27).

Exosome-mediated intercellular communication in osteoarthritis

Exosomes are vital to cell communication because they suppress catabolic factors like MMPs and inflammatory cytokines while boosting anti-inflammatory cytokines, transforming growth factors (TGFs), and other anabolic fac-tors. Exosomes from afflicted locations, such as synovial fluid (SF), OA joints has the reverse impact of healthy exosomes in that they hindered repair enhancement while increasing damage and negative influence in gene expre-ssion. According to a study, healthy chondrocytes were treated with OA-derived exosomes revealed a reduction in the production of anti-inflammatory and anabolic markers. In addition, proinflammatory (M1) macrophages were strongly stimulated by SF-derived exosomes in the release of various inflammatory cytokines, chemokines, and MMPs, but not in the expression of CD80 and CD86 co-stimulatory markers. Furthermore, this study found that SF-derived exosomes enhanced osteoclastogenesis in the absence of macrophage colony stimulating factor as well as receptor activator of nuclear factor kappa-B (NF-κB) ligand (28). In a study, osteoarthritic subchondral bone exosomes activated catabolic genes and decreased chondrocyte specific marker expression. Using RNA sequencing and miRNA profiling, they discovered that miR-210-p was responsible for catabolic gene expression. They were able to suppress miR-210-p using its inhibitor and diminish catabolic gene expression in the OA (29). The elevated expression inflammatory agents eventually target the ECM degrading various types of collagens, proteoglycans (PGs), and other matrix components. Albumin is the most prevalent protein in human blood and a significant component of SF. Albumin content diminishes as the osteochondral defect progresses. Albumin has been reported to have anti-inflammatory characteristics and to upregulate collagen expression during chondrogenic differentiation, which has an impact on the treatment of OA. These findings are still tentative, therefore extensive study into molecular analysis and the mechanism underlying the interaction between exosomes and recipient cells is anticipated.

Exosome as a disease biomarker in osteoarthritis

Exosomes are resistant to degradation, and present in various biofluids, such as blood, SF, and urine, moreover, these characteristics that make them attractive biomarkers for the diagnosis and management of OA. Studies have shown that the content of exosomes in biofluids changes during the progression of OA. These changes include increased levels of proinflammatory cytokines, such as IL-6, and elevated levels of MMPs, break down the ECM. Furthermore, exosomal lncRNAs PCGEM1 that help in the regulation of gene expression and can be used to identify different stages of OA (30). The identification of novel non-invasive biomarkers to inform early diagnosis and disease progno-sis, support personalized treatment selection, and monitor therapeutic progression is of high priority. These biomar-kers can assist medical professionals in making qualified decisions regarding treatment options and fine-tune them as needed to get the best possible outcomes for the patient.

Three-dimensional bioprinting

The field of three-dimensional (3D) bioprinting is rapidly advancing and is widely used for creating tissues and organs. There are three major category of 3D bioprinting based on the technology used: laser-based, inkjet-based, and extrusion-based. Stereolithography is a popular laser-based technology for creating scaffolds with high resolution. Decellularized porcine cartilage ECM is combined with stereolithography to produce a homogeneous bulk distribution of exosomes, which may alleviate chondrocyte mitochondrial dysfunction, promote chondrocyte migration, and trigger M2 macrophage polarization (15). Inkjet bioprinting is a non-contact technique that deposits ink droplets layer by layer and is categorized into piezoelectric and thermal deposition. Inkjet bioprinting was integrated with melt electro writing scaffolds in a recent study (31). Exosomes derived from MSCs have been shown to enhance tissue regeneration and repair in various studies. These exosomes can be incorporated into inkjet bioprinting to enhance the regenerative potential of the printed tissues (32). Extrusion-based bioprinting is a low-cost and convenient method that allows printing of viscous bioinks. On the other hand, the pressure used can result in high shear stress on cells and reduced cell viability. Latest investigation used methacrylated HA hydrogel bioink to 3D print a patch that was effectively infiltrated by human MSCs exosomes, displaying better mechanical performance and high printability. This study demonstrates the potential of exosome-based bioinks for 3D bioprinting applications in tissue engineering and regenerative medicine. This technique has shown outstanding proliferation, migration, and gene expression in fibroblasts and endothelial cells and has been found to be an effective wound healing strategy (33). As revealed by a recent study, exosomes were combined with 3D-printed SF/COL-I/nHA scaffolds to create an elaborate cell-free bone-tissue-engineering system. The scaffolds were lyophilized, crosslinked, sterilized, and retained at −20℃ after being lyophilized and crosslinked. After that, exosomes were inserted into the scaffolds and their release profiles were investigated. The findings revealed that the exosome-loaded scaffolds stimulated angiogenesis and alveolar bone defect healing (34). Similarly, researchers applied 3D printing to construct porous scaffolds and loaded them with bioceramic-induced macrophage exosomes. The results showed that the exosome-loaded scaffolds had immunomodulatory effects and promoted osteogenesis and angiogenesis (35). Likewise, researchers developed a “cell-free scaffold engineering” strategy that integrates strontium and highly bioactive sEXOs inside a 3D-printed scaffold. The results showed that the sEXO-loaded scaffolds had enhanced osteogenesis and angiogenesis, making them a promising approach for critical bone defects (36). Recently, researchers developed a novel bioinspired double-network hydrogel scaffold produced via 3D printing with tissue-specific decellularized extracellular matrix (dECM) and exosomes. The results showed that the exosome-reinforced hydrogel scaffolds had efficient cartilage and subchondral bone regeneration (37). Overall, the use of exosomes in 3D bioprinting is a promising area of research that holds great potential for the development of novel therapeutic approaches for tissue engineering and regenerative medicine.

Biomaterials

In the field of bioprinting, natural biomaterials such as hyaluronate, gelatin, collagen, alginate, and dECM, or synthetically produced polymer composites like polylactic acid, polyglycolic acid, polycaprolactone, and poly lactic-co-glycolic acid (PLGA) are commonly used. Inorganic components such as graphene, β-tricalcium phosphate, and nanoparticles are coupled with scaffolds to enhance their mechanical properties. Hydrogels are capable of absorbing water and entrapping necessary components, are currently being applied in tissue regeneration owing to their high-water content, crosslinking processes, and ability to function as a 3D hydrophilic polymer matrix. Hydrogels made of natural biopolymers like alginate, chitosan, gelatin, and HA, as well as synthetic biopolymers such as PEG, PLGA, and poly(2-hydroxyethyl methacrylate). Exosomes, when combined with biomaterials, hold great potential for the development of novel therapeutic approaches for tissue engineering and regenerative medicine. Recent studies have shown that exosome-laden injectable hydrogels can improve diabetic wound healing, promote macrophage polarization, and accelerate angiogenesis. Xing et al. (38), created an injectable thermosensitive hydrogel using ADSC generated exosomes and dECM hydrogel. Furthermore, synthetic poly(D,L-lactide)-block-poly(ethylene glycol)-block-poly(D,L-lactide) triblock copolymer gels have demonstrated potential for cartilage preservation and slowing the advancement of OA. On the other hand, hydrogel was prepared by combining exosomes with polymers and crosslinkers for gelation. The study found that the GMOCS hydrogel significantly promoted the synthesis of ECM due to the doping of OCS. Additionally, the combination of BMSC-derived exosomes with a hydrogel scaffold can improve its bioactivity, which can help human umbilical vein endothelial cells grow (39). In a study, fabricated GelMA/nano clay/sEVs hydrogel was crosslinked with ultraviolet light and Laponite nano clay was added to improve the biological and mechanical properties and to release sEVs in a sustained manner with hydrogel degradation (40). On the other hand, PLGA–PEG–PLGA thermogel combined with kartogenin was used as an intraarticular injection and was shown to improve cartilage repair in an anterior cruciate ligament transection surgery-induced OA model (41). According to recent research, sEVs produced from synovial mesenchymal stem cells can enhance chondrocyte proliferation and migration. circRNA3503-OE-sEVs alleviated inflammation-induced apoptosis and the imbalance between ECM synthesis and ECM degradation by acting as a sponge for I-miR-181c-3p and hsa-let-7b-3p and was considerably elevated following melatonin-induced cell sleep. The researchers looked at the viability of using sEVs in conjunction with sleep-related circRNA3503 as a targeted therapeutic agent using injectable thermosensitive hydrogel to prevent OA. PDLLA-PEG-PDLLA; PLEL triblock copolymer-based gels were applied in the investigation, which gradually released circRNA3503-loaded sEVs. The researchers discovered that circRNA3503-OE-sEVs diminished inflammation-induced apoptosis and the imbalance between ECM production and degradation via numerous paths as a result, that sEVs and circRNA3503 have the potential to be used as a targeted treatment agent with injectable thermosensitive hydrogel to prevent OA development (42). Additionally, AD/CS/RSF hydrogel has been found to promote BMSC migration, proliferation, and differentiation in cartilage defect regeneration and remodelling of the ECM. In animal models of cartilage damage, the AD/CS/RSF/EXO hydrogel with encapsulated exosomes has been found to facilitate BMSCs migration and inflation, stimulate BMSCs proliferation and differentiation, and enhance the therapeutic efficacy of MSCs-exosomes (43). In animal models of cartilage damage, the hydrogel sponges render MSCs-exosomes more stable in vivo and expedite tissue healing and regeneration. Ultimately, the utilization of exosomes in combination with biomaterials is an exciting area of research that holds the potential to pave the way to the development of innovative therapeutic techniques for tissue engineering and regenerative medicine.

Nanoparticles

Some research has shown that using special nanoparticles to improve the exosomes biological characteristics could be beneficial. Although this strategy was not utilized in the treatment of OA or cartilage injury. The nanoparticles can prevent the release of exosomes. The mechanism by which these nanoparticles can prevent the exosomes from releasing is related to the silencing of genes. Researchers found that exposure to silica nanoparticles can decrease the exosomes secretion. According to a study conducted by Khongkow et al. (44), the ability of nanoparticles to enhance exosomes targeting ability was improved by using a mechanical method or by exosomes derived from healthy human cells. It is advantageous to improve the drug loading capacity of exosomes in order to broaden the spectrum of medication selection and locate the optimal medicine for OA treatment/cartilage repair.

Interactions between exosomes and chondrocytes

Exosomes have been found to play a crucial role in maintaining chondrocyte density, promoting cell proliferation and differentiation, and inhibiting excessive chondrocyte death. In a surgically induced rat model of knee OA, intraarticular injection of bone marrow mesenchymal stem cell-derived exosomes (BMSC-EXOs) was shown to alleviate cartilage injury and modulate subchondral bone remodelling (45). In vitro experiments revealed that BMSCs-derived exosomal lncRNA MEG-3 increased collagen type II (COL II) synthesis, while inhibiting chondrocyte senescence and apoptosis induced by IL-1β. BMSC-EXOs also ameliorated IL-1β-induced inhibition of chondrocyte proliferation and migration. The upregulation of COL II and aggrecan, as well as the down-regulation of MMP13 and ADAMTS5, were further confirmed in vivo (46). Addi-tionally, exosomes have shown to activate autophagy in various diseases, including OA. BMSC-EXOs have been found to regulate the PI3K/AKT/mTOR signalling axis in IL-1β-induced degenerative disc disease, activate autophagy, inhibit the release of inflammatory mediators, and reduce excessive annulus fibrosus cell apoptosis (47). The mutually promoting and positive cycle between exosomes and chondrocytes highlights the potential therapeutic applications of exosomes in OA.

Exosomes contribution to cartilage degradation and inflammation

Chondrocytes that dwell in an ECM mesh that constitute cartilage. The ECM is composed primarily of the network COL II and an interlocking mesh of fibrous proteins and PGs, HA, and chondroitin sulfate (CS). When chondrocytes and ECM are in a healthy state, they work together to sustain a balance between synthesis and degradation. In pathological conditions such as OA, there is an increase in ECM degradation, resulting in decreased collagen and proteoglycan levels. Exosomes have been shown to reduce cartilage damage and ECM degradation by increasing COL II expression and decreasing ADAMTS5 expression in a rat model of OA (48). Similarly, BMSC-EXOs also showed promise in promoting the expression of COL II and aggrecan while inhibiting catabolic enzymes and protecting chondrocytes from apoptosis. Exosomal miR-125a-5p and miR-136-5p were found to attenuate ECM degradation by targeting E2F2 and ELF3, respectively, in a mouse model of traumatic OA (49). Exosomes diminish the activity of inflammatory mediators, which is the requisite for treating inflammation, and aid in the conservancy of cartilage without its disintegration. The use of exosomes along with biomaterials is a promising area of research that holds great potential for the development of novel therapeutic approaches for tissue engineering and regenerative medicine.

Exosomes in promoting angiogenesis and fibrosis in osteoarthritis

Exosomes have been found to promote angiogenesis in OA by stimulating the growth of new blood vessels known as neovascularization, considered to contribute to the progression of the disease by alleviating the supply of oxygen and nutrients to the affected joint. Exosomes can promote neovascularization by liberating pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor. Recent research has provided updated insights on the pathogenesis, diagnosis, and treatment of OA using exosomes derived from annular fibrous cells reduced angiogenesis in intravertebral disc degeneration by downregulating VEGF (50).

In addition to promoting angiogenesis, exosomes also play a role in the development of fibrosis in OA. Fibrosis is the accumulation of scar tissue in the joint, which can contribute to cartilage loss and joint dysfunction. Exosomes can cause fibrosis by stimulating fibroblasts, the cells responsible for the production of collagen and other ECM components, that in turn mediated by the release of TGF-β and other pro-fibrotic factors. The lack of angiogenesis regulation in the pathogenesis of OA can potentially be addressed by establishing a feedback mechanism using exosomes, which could retard the disease progression. Whereas, the role of angiogenesis in the progression of OA is in dispute, and further exploration on angiogenesis-based treatments should be interpreted carefully. Additionally, the debate on whether cartilage repair can be promoted without exacerbating inflammation and pain still underway. The regulation of angiogenesis by exosomes presents area of concern, as the underlying mechanisms are still not well understood. A comprehensive understanding of exosome-mediated angiogenesis in OA could lead to significant therapeutic advancements.

While current treatments for OA, such as HA and Platelet Rich Plasma therapy, have shown some benefits, they may not be effective enough for everyone. Exosomes, on the other hand, have emerged as a promising alternative for the treatment of OA. Exosomes have the ability to suppress catabolic factors and boost anti-inflammatory cytokines, making them a potential tool for the treatment of OA (Fig. 2). Fig. 2 discusses the clinical applications of exosomes. Exosomes have shown great potential for the treatment of various diseases, and many preclinical experiments have confirmed their advantages in the field of regenerative medicine. For instance, exosomes can be administered directly to the joint space through intra-articular injection. Studies has proven that direct introduction of exosomes to the joint decrease pain and inflammation and promote the tissue repair furthermore, exosomes can also be administered systemically, such as through intravenous injection (51). Specifically, systemic administration of exosomes may provide a broader distribution of the therapy and decreased levels of proinflammatory cytokines, apoptotic proteins and TLR4/NF-κB signalling (52). Exosomes can also be utilized in combination with other therapies, such as platelet-rich plasma or HA injections. Combination therapy, which involves the use of two or more therapeutic agents, has been shown to be more effective than individual therapies alone. This approach can lead to additive or synergistic effects, enhancing the therapeutic effects of each individual therapy. Nonetheless, exosomes derived from ADSCs have become a hot topic in the field of personalized medicine, and their potential applications in various fields are being explored (53). This approach may reduce the risk of immune rejection and improve treatment efficacy.

Figure 2. Application of exosomes. Schematic representation of the potential applications of exosomes in wound healing, chondral defect repair, anticancer drug delivery. Exosomes can be loaded with endogenous cargos or intentionally changed molecules in a variety of ways, and many exosome delivery routes for treatment have been developed. Exosomes, on the other hand, require targeted tactics to boost medication efficacy. Exosomes generated from stem cells have been discovered to offer therapeutic promise for a wide range of injuries and disorders, including bone regeneration and wound repair.

Clinical trials are currently underway to evaluate the safety and efficacy of exosome therapy in OA. The clinical trials of Exosomes for Malignant and Acute Conditions conducted to date are listed in the (Table 3). If these trials demonstrate positive results, exosome therapy may become a viable treatment option for patients with OA and other conditions.

Table 3 . Clinical trials of exosomes for malignant and acute conditions

Disease	Clinical trial ID	Study title	Phase	No. of patients	Study type	Source of MSC	Route of administration	Dose
OA	NCT05060107	Intra-articular injection of MSC-derived exosomes in knee OA	1	10	Open label	N/A	IA	Single dose (3∼5×1011 particles/dose)
Macular degeneration	NCT03437759	MSC-Exos promote healing of MHs	1	N/A	Randomized	Umbilical cord tissue	Local	50 or 20 μg MSC-Exos
Type I diabetes mellitus	NCT02138331	Effect of microvesicles and exosomes therapy onβ-cell mass in type I diabetesmellitus (T1D)	02-Mar	N/A	Interventional open-label	Umbilical cord blood	IV	Two doses (one week apart) of exosomes produced from 1.22∼1.51×106 MSCs/kg
Ischemic stroke	NCT03384433	Allogeneic mesenchymal stem cell-derived exosome inpatients with acute ischemic stroke	01-Feb	5	Randomized, single-blinded, placebo-controlled	Bone marrow	Intraparenchymal	Single dose of (200 mg MSC-Exos)
Dry eye	NCT04213248	Effect of UC-MSC-Exos on dry eye in patients With cGVHD	01-Feb	27	Single group	Umbilical cord	Artificial tears	UC-MSC-Exos 10 μg/drop, 4 times a day for 14 days
Alzheimer’s disease	NCT04388982	The safety and the efficacy evaluation of allogeneic adipose MSC-Exos in patients with Alzheimer’s disease	01-Feb	9	Non-randomized	Adipose tissue	Nasal drip	5/10/20 μg ASC-Exos weekly twice
ARDS	NCT04602104	A clinicalstudy of mesenchymal stem cell exosomes nebulizer for the treatment of ARDS	01-Feb	169	Randomized, double-blinded, controlled	N/A	Inhalation	2.0×108 exosomes, 8.0×108 vesicles, 16.0×108 exosomes (every day in a week)
Pulmonary infection	NCT04544215	A clinical study of mesenchymal progenitor cell exosomes nebulizer for the treatment of pulmonary infection	01-Feb	60	Randomized, double-blinded, controlled	Adipose tissue	Inhalation	8.0×108 exosomes/3 ml, 16.0×108 exosomes/3 ml (every day in a week)
Dystrophic epidermolysis bullosa	NCT04173650	MSC-EVs in dystrophic epidermolysis bullosa	N/A	10	Single group	Bone marrow	Local	N/A
Multiple organ dysfunction syndrome	NCT04356300	Multiple organ dysfunction syndrome after surgical repair of acute type A aortic dissection	N/A	60	Interventional	N/A	IV	150 mg once a day (14 times)
Periodontitis	NCT04270006	Evaluation of adipose derived stem cells exo in treatment of periodontitis (exosomes)	1	10	Open label	Adipose tissue	Local	N/A
Metastatic pancreas cancer	NCT03608631	iExosomes in treating participants with metastatic pancreas cancer with KrasG12D mutation	1	28	Open label	N/A	IV	N/A
COVID-19	NCT04276987	A pilot clinical study on inhalation of mesenchymal stem cells exosomes treating severe novel coronavirus pneumonia	1	24	Pilot/open-label	Adipose tissue	Inhalation	5 times of MSC-Exos (2×108 nanovesicles/ 3 ml at Day 1∼5)
SARS-CoV-2	NCT04491240	SARS-CoV-2 associated pneumonia	1	30	Interventional	N/A	Inhalation	Twice a day for 10 days inhalation of 3 ml (0.5∼2×1010 of nanoparticles)
Normal and colon cancer tissue	NCT01294072	Phase I clinical trial investigating the ability of plant exosomes to deliver curcumin to normal and malignant colon tissue	1	35	Interventional	Plant (grape)	Curcumin cargo	Curcumin combined with plant exosomes (every day in a week)

MSC: mesenchymal stem cell, ARDS: acute respiratory distress syndrome, COVID-19: coronavirus disease 2019, SARS-CoV-2: severe acute respiratory syndrome coronavirus 2, OA: osteoarthritis, N/A: not available, IA: intraarticular, MSC-Exos: MSC-derived exosomes, MHs: medication history, IV: intravenous, UC: ulcerative colitis, cGVHD: chronic graft-versus-host disease, ASC: adipose stem cell, EVs: extracellular vesicles.

Exosomes have the potential to regenerate damaged cartilage. In this review, we have summarized current research on therapies for cartilage regeneration, with a focus on OA. We have highlighted studies that investigate various cell-derived exosomes. Multiple in vivo and in vitro investigations have implicated exosomes in the repair of cartilage defects (54). While the evidence for their efficacy in treating cartilage-related issues is convincing, several considerations should be taken into account before proceeding with clinical trials. To ensure accurate results, it is important to use large animal models in studies, as small animals do not yield positive outcomes when assessing cartilage thickness in relation to prenatal and postnatal influences. Additionally, the optimal dosage of exosomes needs to be determined, and more research is required to identify the active components within exosomes. Further studies should be conducted using exosomes from larger donor cohorts to establish their efficacy. Since exosomes are complex carriers containing DNA, RNA, lipids, metabolites, and various proteins, they exhibit therapeutic functions through immunomodulatory, bioenergetics, or biochemical effects in various disease models. Therefore, it is crucial to explore the underlying mechanisms of exosome therapy in

OA further. Moreover, the long-term safety of exosome therapy in OA remains unknown, necessitating comprehensive studies to evaluate potential side effects. Animal model studies should also extend their observation time points to provide a more accurate reference for the application of exosomes in treating OA. Exosome therapy for OA is currently considered an experimental treatment and has not yet received regulatory approval from agencies such as the U.S. Food and Drug Administration. Regulatory approval is essential to ensure the safety and efficacy of this therapy. Considering that exosomes have a short half-life in the circulatory system, utilizing a hydrogel scaffold can be a suitable alternative for targeted delivery of exosomes. However, hydrogel delivery faces limitations such as non-effective crosslinking, the use of strong organic solvents for degradation, and exosome adhesion issues due to the absence of an ECM-like environment. Utilizing 3D bioprinting technology can help create more suitable culture conditions for exosomes and improve scaffolding for their delivery.

In conclusion, exosomes play a vital role in intercellular communication and contribute to the regeneration of cartilage-associated defects, particularly in OA. Extensive research has been conducted on the potential therapeutic targets of exosomes in treating chondral defects and OA. However, the optimization of exosomes is necessary to advance their therapeutic applications. Before exosomes can be effectively used in clinical practice, several challenges still need to be addressed. Although studies on the roles, properties, and applications of exosomes are in their early stages, recent research has shown promising results in utilizing exosomes for cartilage repair and regeneration. Tissue engineering techniques, such as hydrogels and scaffolds, have been employed to enhance the localization of exosomes for targeted drug delivery to specific cartilage defects. The combination of exosomes and hydrogels has demonstrated potential in stimulating chondrocyte proliferation and migration, reducing inflammation-induced apoptosis, as well as balancing the formation and degradation of cartilage matrix. With further research aimed at optimizing their therapeutic potential, exosome-based therapies for chondriodefect regeneration are expected to gain prominence in the near future.

There is no potential conflict of interest to declare.

Conceptualization: JS, SCL, JKS. Data curation: JS. Formal analysis: JS. Funding acquisition: JKS. Methodology: JS. Project administration: JKS. Supervision: SCL, HKK, JKS, JYK. Visualization: SSK. Writing – original draft: JS. Writing – review and editing: JS.

This work was supported by the Korea Fund for Rege-nerative Medicine (KFRM) grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Health & Welfare) (22C0603L1-11).